From Production Skills to Scientific Knowledge,
and Onward to Fusion with Medical Science

TMR-MTA cement Mielle

Product ReportFeature of TMR-MTA cement Mielle and key point of treatment

Made inJapanh
BismutFree

Edited by YAMAKIN Ph.D. Group1

1

Introduction
We have been actively engaging in industry-academia collaborations with universities and research
institutions to advance the research and development of dental materials. This "industry-academia
collaboration" has allowed for innovation in advanced technology to be achieved through technologicalinteractions with universities and other research institutions.
In 2011, the YAMAKIN Ph.D. Group, was established as an internal expert organization, consisting ofdoctoral degree-holders in dentistry, pharmacy, engineering, science, agriculture, and Entrepreneurial
Engineering. Regardless of the members’ company history or field of work, the Group is the driving force
to continually generate innovation by integrating knowledge, experience, and technology across multiple
disciplines rather than working in isolated specialist fields.

The story of the MTA Cement Project:
Prof. Kazuhiko Endo Ph.D. (Division of Biomaterials and Bioengineering, Department of Oral
Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido) and Prof. Masato Saito
Ph.D. (Division of Pediatric Dentistry, School of Dentistry, Health Sciences University of Hokkaido)
were conducting basic studies on MTA cement., The YAMAKIN agreed with the ideas of Professors
Endo and Saito, and launched the MTA Cement Project in spring 2012. The fundamental research isvery much done by the experts at Health Sciences University of Hokkaido. It has progressed at a rapid
pace, and the knowledge base has expanded continuously.
As a result, we succeeded in finding an outstanding composition in 2014.
However, MTA cement is not a simple material that can be completed on an as-is basis when its
constituent powders are mixed. Differences in manufacturing conditions greatly affect the performance
underlying MTA cement , such as setting time and compressive strength.
It is also necessary to strictly control production environment factors such as humidity andcleanliness, given the product’s direct contact with the pulp. Each of these challenges has been
overcome, and in July 2017, we were able to release our conventional product, TMR-MTA Cement, forcommercialization.

TMR-MTA cement Mielle, a product that responds to the opinions of clinical practitioners
Our conventional product, TMR-MTA Cement has been well received for its superior operating,physical, chemical, biological, and aesthetic properties, but there have been requests from clinicalpractitioners to improve the X-ray visibility without sacrificing the merits of the conventional product.
In order to respond to such opinions, a new project was started immediately, leading to the launch of
TMR-MTA cement Mielle two years after the release of the conventional product.
This report covers the characteristics and usage of the TMR-MTA cement Mielle and offers access tovideo for areas that are difficult to image with text and photographs alone. Please read the QR code
attached. Dental treatment continues to change tremendously in its technologies and systems. We willcontinue to strive for the development of the dental industry by creating new products that meet theneeds of the times through further innovation.

2

Supervisors:
YAMAKIN Ph.D. group
Dr. Teruo Anraku (Ph.D. in Engineering)
Dr. Hiroyuki Itoigawa (Ph.D. in Science)
Dr. Takahiro Kato (Ph.D. in Engineering)
Dr. Takeshi Sakamoto (Ph.D. in Pharmaceutical Science)
Dr. Yuji Sato (Ph.D. in Entrepreneurial engineering)
Dr. Hidekazu Tanaka (Ph.D. in Engineering)
Dr. Ritaro Matsuura (Ph.D. in Agriculture)
Dr. Masatoshi Yamazoe (Ph.D. in Dentistry)Dr. Hirohisa Yamamoto (Ph.D. in Entrepreneurial Engineering)
Advisor of YAMAKIN Ph.D. group
Dr. Bunichiro Yamada (Ph.D. in Engineering)

What is YAMAKIN Ph.D. group?
This is an expert group from various specialties in YAMAKIN, and it is the driving force to continually
generate innovation by integrating diverse knowledge, experience, and technology.

3

2

What is MTA cement?
If the pulp is exposed due to caries treatment or other such intervention, pulp protection is performedby sealing the pulp with dental material to protect the nerve (Figure 2-1). MTA cement is a dental
pulp-capping material used in this treatment. The MTA in the name stands for "Mineral Trioxide
Aggregate" and, in MTA cement, materials that confer X-ray contrast, such as bismuth oxide, are added
to the mineral trioxide aggregate. Generally speaking, “cement” in dentistry is strongly associated withglass ionomer cement and adhesive resin cement, so there are some people who misunderstand MTA
cement as being used for coalescence and adhesion. However, the term "cement" is used because thecalcium silicate-based raw materials that are the main component of MTA cement are close to Portland
cement, used for building materials.
The launch of MTA cement in the Japanese market began in April 2007 with "PROROOT MTA" soldby Dentsply Sankin Co., Ltd. (now Dentsply Sirona). In Japan, it is approved only for use as a pulpcapping material for dental use, but overseas it is applied not only for indirect or direct pulp capping,but also in various clinical applications, such as closure of perforations, root canal filling, and reverseroot canal filling material for root-end resection.
Caries-affected area

Exposed pulp

MTA cement

Dentalpulp

Removal of caries-affected areas

MTA cement filling

Figure 2-1 Schematic illustration of the use of MTA cement (pulp-capping)2-1 Characteristics of MTA cement
The merits of treatment using MTA cement are summarized in Table 2-1. The operational method isvery easy: simply make the material paste by mixing powder and water, and cap the exposed pulp.
Table 1 The Merits of Treatment Using MTA cement●Setting by Mixing with Water
MTA cement is set by being mixed with water,
making it suitable for use in the intraoral area.

●Biocompatibility(Effect of Promoting Hard Tissue Formation)
The cytotoxicity of MTA cement is lower than that ofother dental cements, and MTA cement promotes
the formation of new dentine.

●Regenerating Tooth Material
MTA cement promotes the formation ofhydroxyapatite, which is a constituent of teeth,by releasing CA2+ at the dentinal tubules.

4●Sealing Ability
As MTA cement expands slightly while it is set,
restrains leakages at the microscopic level are
inhibited, preventing bacterial infection.

●Disinfectant Ability
MTA cement shows strong alkalinity (pH 12) during
the setting process, it disinfects the adhering andsurrounding bacteria.

● Preserving Dental Nerves
MTA cement enables treatment to proceed leaving
nerves undisturbed, extending the life of teeth.

After setting, MTA cement prevents the recurrence of caries by its sealing ability; moreover, it detersbacteria and demonstrates antibacterial ability caused by strong alkalinity of calcium hydroxide, whichis generated by reacting with water. (Figure 2-2) Also, calcium ion in the component is released thenhydroxyapatite is formed around it; moreover, the formation of new dentine will be promoted by thehigh biocompatibility.

2 (3CaO・SiO2)+

Tricalcium Silicate

2 (2CaO・SiO2)
Dicalcium Silicate

6H2O

→

Water

+

4H2O
Water

3CaO・2SiO2・3H2O+

Calcium Silicate Hydrate→

3CaO・2SiO2・3H2O
Calcium Silicate Hydrate

3Ca(OH)2
Calcium Hydroxide+

Ca(OH)2
Calcium Hydroxide

Shows antibacterial ability caused by strong alkalinity

Figure 2-2 Hydration Reaction of MTA cement
Thanks to these effects, nerves can be protected even with pulp exposure; as a result, treatment can bedone preserving the pulp as much as possible. Especially in the field of pediatric dentistry, there is astrong need for MTA cement as pulpectomy will affect the formation of permanent tooth substance.

52-2 Mechanism of Setting Reaction
The mechanism of setting reaction by mixing MTA cement and water is as follows:
1) Hydration reaction occurs on surfaces of cement particles and hydrate crystals are extracted on thesurfaces.
2) Setting reaction starts slowly as hydrate crystals on the surfaces of cement and surrounding hydrate
crystals become interlocked.
Regarding the hydration reaction which is shown in Figure2-3, if the amount of water is insufficient,the density of hydrate crystals becomes low and coupling between particles will be weaker; as a result,setting will be insufficient 1). However, if too much water is added, the excess water, which will notaffect the hydration reaction, will lower the compressive strength, because the number of spaces inside
the cement will increase; therefore, avoid adding too much water 1). Also, the particle diameter andsurface area differ depending on the product; so, the moisture ratio has to be adjusted in each case,
based on the recommended moisture ratio given by the manufacturer.

Bonding water• In cases where
moisture is sufficient

Excess water
Cementparticle

Cementparticle

Bonding water for hydration reaction
will adhere to a cement particle.

Hydrate crystals are generated by thehydration reaction of the bonding
water and a cement particle.

Cementparticle

Cementparticle

The bonding water evaporates

The number of hydrate crystals decreases• In cases where
the moisture is insufficient

Bonding water

The low number of hydrate crystalscauses lowering of the strength.

Excess water
Cementparticle

Cementparticle

There will be more excess water thanrequired bonding water forhydration reaction.

Hydrate crystals are generated ona cement particle by the hydration
reaction of the bonding water.

• In case where
the moisture is excessive

The strength will be exhibitedby the hydrate crystals’ being
dense and interlocked with each other.

Because of the excess water, space of thecement particles becomes wider, andentanglement of the hydrated crystals will
be weaker, thus the setting process takes
longer. Also, the strength is greatly reduced.

Figure 2-3 The Difference in Hydration Reaction Caused by the Amount of Water

6

2-3 Comparison with calcium hydroxide preparations
Generally, calcium hydroxide preparation is used for pulp protection procedures. It exhibits hardtissue-inducing action and disinfectant ability caused by its strong alkalinity; moreover, the material
cost is low. Therefore, it is widely used in various endodontic treatments. However, unlike MTA
cement, calcium hydroxide preparations do not harden (set), so that the strength of the sealing is
weak 4), and the components are easily eluted. Its elution further reduces sealing ability, and thus, it
can be said that there is a high risk of bacterial infection in the long term. However, the lack of ability
to set is a merit when the purpose is to sterilize the root canal because it needs to be removed at thetime of re-examination.
In the comparisons of the long-term outcomes of pulp protection with MTA cement and calcium
hydroxide preparations 5), the MTA cement success rate* was approximately 80% or more after three
years, whereas the success rate after one year with calcium hydroxide preparations was approximately
75%, which is roughly equal to MTA cement. However, the success rate falls to 55% after two years andto about 45% after three years. *“Success rate” is evaluated from clinical findings on the presence orabsence of pain, (e.g., spontaneous pain, occlusal pain, percussion pain, cold/warm water pain), toothsway, periodontal pocket confirmation, and radiographic findings of the root canal segments at reexamination.
In conservative treatment, long-term stability is considered essential. Therefore, MTA cement is avaluable material for patients.

References
1) Mahmoud Torabinejad (edited), Yoshitsugu TERAUCHI (inspected): complete MTA. Quintessence Publishing, 2017.
2) Eldeniz AU, Hadimli HH, Ataoglu H, Orstavik D: Antibacterial Effect of Selected Root-End Filling Materials. J Endod, 32(4), 345-349, 2006.
3) Takashi OKIJI (Edited): MTA its basics and clinics. Hyoron Publishers, Tokyo, 2016.
4) Horsted-Bindslev P, Lovshall H: Treatment outcome of vital pulp treatment. Endod Topics, 2, 24-34, 2002.
5) Mente J, Geletneky B, Ohle M, et al.: Mineral trioxide aggregate or calcium hydroxide direct pulp capping: an analysis of the clinical treatment outcome.
J Endod, 36(5), 806-813, 2010.

7

3

Features of TMR-MTA cement Mielle

3-1 Usability
TMR-MTA cement Mielle is characterized by the fact that when water comes into contact with thecement, it is quickly absorbed and turns into cement mud that can be easily kneaded (see Video 2).
This is because the presence of zirconia particles and spherical silica particles between the calcium
silicate particles creates small spaces between the calcium silicate particles, and water is absorbed by
the capillary phenomenon. Besides, the bearing effect (Figure 3-1) of the spherical-silica fine particles
improves the product’s fluidity. This enables users to make an even paste easily with a small amount ofwater in a short time.

Calcium
silica particle
Spherical-silica
fine particle
Zirconia
fine particle

Improving fluidity
Image diagram of bearing effectBall bearing

Figure 3-1 Image of Bearing Effect

3-2 Setting Time and Self-Setting Ability
3-2-1 Initial setting time
As discussed above, TMR-MTA cement Mielle mixes well to an even paste with a small amount ofwater. Since it can be mixed with a small amount of water, its designed setting time is short, so that theinitial setting is completed in 15 to 30 min. when the moisture ratio is 20%. In this section, the effectof moisture ratio during mixing and initial setting time of MTA cement are evaluated.
Materials and Methods
The initial setting time was measured in accordance with themeasurement of setting time in ISO 6876:2012 and JIS T 6522:2015(Dental Root Canal Sealing Materials). That is, paste-like samples
obtained by mixing MTA powder and distilled water were filled into
molds with a diameter of 10 mm and a depth of 2 mm (Figure 3-2),
and a load was applied by a Vicat needle (mass: 100.0 ± 0.5 g, tip
diameter: 2.0 ± 0.1 mm plane, Figure 3-3) while measuring the timefrom the time of mixing, and the setting time required to obtainhardness at which no trace of the Vicat needle appeared on thesurface was taken as the initial setting time.
The moisture ratio at the time of mixing the MTA powder and thedistilled water was adjusted using a micropipette.

Figure 3-2 Sclerotic test sample

Figure 3-3 Vicat needle tester8

Results and Discussion
As shown in Figure 3-4, the time required for setting tended to increase with increasing water ratio.
When the moisture ratio is 35% and 40% by mass (about 0.108 g and 0.133 g of distilled water to 0.2 g
of MTA powder, respectively), the paste form cannot be maintained after mixing because the wateramount is excessive.
In this case as well, although it is set until finally no trace of the needle appears, the compressive
strength described below is lowered, so that it is necessary to remove excessive moisture.
By adjusting the appropriate amount of moisture with dry cotton or the like, the initial setting time
can be shortened.
However, this initial setting time is the time taken until the cement component sets to some extent,
and corresponds to the time called solidification time in industrial cement 1), not to the time when
the cement component completely sets due to the hydration reaction. Since the hydration reaction
progresses fully after initial setting, and the strength increases, the measurement of compressive
strength was measured 1-7 days after adequate setting had progressed.
350

Initial setting time (min)

300
250
200
150
100
50
0

0

10

20

30

40

50

Moisture ratio (%)

Figure 3-4 Relationship between moisture ratio and initial setting time

3-2-2 Collapsibility in water before initial setting
If MTA cement is dissolved or eluted before initial setting by contact with body fluids, there are concernsthat performance of MTA cement will be impaired. Therefore, in this section, the collapsibility ofMielle in water before initial setting was evaluated.
Materials and Methods
MTA cement
Pasted samples of Mielle and distilled water mixed at 20%
moisture ratio were filled into a 1 mm-diameter acrylic
Acrylic board
hole of 2 mm depth and immersed in 37°C distilled water
immediately after filling (Figure 3-5). As a control sample,
calcium hydroxide powder was used mixed with distilledwater at a moisture ratio of 50% so as to have a paste
viscosity equivalent to that of Mielle. The samples were
Observation of the spillage andsurface condition of the sample
removed from the water after 24 h to check the surface
Figure 3-5 Diagram of disintegration test
condition. In addition, the amount of decrease (mm) fromthe exposed surface of the sample before immersion was measured using a micrometer, and the amountof dissolution/outflow was determined.

9

Results and Discussion
As shown in Figure 3-6 and Figure 3-7, no elution or collapse of the surface was observed in Mielle. On
the other hand, in calcium hydroxide, dents were observed on the surface, and elution and collapse of
the sample were observed.
No change

MielleMielleCalcium hydroxide

In water at 37 ˚C Slight depression
for 24 hours

Figure 3-6 Changes in appearance
before and after immersion in water

Decrease in volume (mm)0.000

Calcium hydroxide

0-0.5000.677-1.000-1.500-2.000Figure 3-7 Elution of Samples in Water Immersion

As a result of this test, it was confirmed that if water pressure for washing with a three-way syringe,
etc. is not applied to Mielle, it will not dissolve or flow out in water even immediately after filling.

3-3 Compressive strength
The strength of MTA cement after setting is assessed by compressive strength. In this section, thecompressive strength of Mielle was tested in accordance with JIS T 6609-1:2005 (Dental Water-Based
Cement-Part 1: Powder/liquid Acid-base Cements) to determine the effect of setting conditions oncompressive strength.
MATERIALS AND METHODSColumnar samples (Figure 3-8) were preparedby filling stainless-steel split jigs with internal
dimensions of 4 mm in diameter and 6 mm
in height, with samples obtained by mixing
MTA powder samples with 1) distilled waterat a moisture rate of 20% by mass (0.1 g ofdistilled water to 0.4 g of powder) and 2) water
ratio of 18, 20, 23, 25, 30, and 35% by mass.

Figure 3-8
Figure 3-9 Compact Universal
Compressed specimen
Testing Machine

Thereafter, a compressive load was applied using a small universal tester (EZ-Graph: Shimadzu
Corporation, Figure 3-9). (1) a sample stored for 24 hours in water at 37°C, and a sample stored in air
with a humidity of 90% or more for 24 hours. This test shows the difference between the condition inwater and in air.
(2) a sample stored in water at 37°C for 24 hours and a sample stored in water at 37°C for 168 hours (7days). This test shows the difference between the condition in water after one day and after seven days.
The compressive strength of each above samples were calculated from the load when the sample wasbroken.

10

Results and Discussion
Figure 3-10 shows the compressive strength of Mielle when it is mixed at a moisture ratio of 20 mass%.
MTA cement is commonly used for treatment in which a wet cotton swab is placed after filling MTA
cement and then a composite resin is filled on the next day or later.
Mielle stored in air demonstrates compressive strength equivalent to that of a sample stored in water(assuming a wet cotton swab after MTA cement is filled). In this way, Mielle is sufficiently set in thesame way as the conventional product, with no need for moisture replenishment by moist cotton swabs,
etc. Thus, it enables a one-off filling of the composite after MTA cement Mielle has been used. Bothprocedures are performed on the same day, which reduces the burden on patients.

Compressive Strength (MPa)

120

90

60

30

0

Storage in water

Storage in air

Figure 3-10 Compressive strength of Mielle(1 day after filling; water ratio of 20 mass%)
In addition, the results for compressive strength compared to the conventional product are shown in
Figure 3-11. Since setting time of Mielle is as fast as that of the conventional product, it shows highcompressive strength (about 90 MPa) at 20 mass% moisture ratio after one day of settlement, and it
increases to about 140 MPa after one week. This is a compressive strength comparable to that of glassionomer cement, which is sufficient as the strength required for pulp protection.

Compressive strength (MPa)Conventional product

Mielle

160

120

80

40

0

One day after filling

1 week after filling

Figure 3-11 Compressive Strength of the Conventional Product and Mielle
Figure 3-12 shows the compressive strength of Mielle when it is mixed at various moisture rates. The
material properties of MTA cement are known to become higher when the moisture ratio decreases andlower when the moisture ratio increases 3). The result of this test similarly showed that its compressive
strength of Mielle tended to be higher when moisture ratio was low and lower when water ratio washigh 1 day and 1 week after filling, respectively 4).

11Compressive strength (MPa)200

1 day after filling with Mielle

1 week after filling with Mielle

150

100

50

0
18

20

23

25

30

35

Moisture ratio (mass%)

Figure 3-12 Compressive Strength of Mielle by Moisture Ratio
When the water amount at the time of mixing was 30% by mass or more (0.085 g or more of water to
0.2 g of powder), the water ratio of the mixed product became excessive and the strength after setting
was insufficient. This may be attributed to the fact that, as shown in Figure 3-13, the gap between the
cement particles increases in the presence of excessive moisture, so that although hydrated crystals
grow and harden over time, but sufficient adhesion strength cannot be obtained.
Since Mielle can achieve a uniform paste even with a small amount of water due to the bearing effectof spherical particles, water should be added little by little in small amounts while mixing, so that theamount of water is appropriate. If the moisture becomes excessive, it is necessary to absorb the excess
moisture with dry cotton or the like before filling.
Water: Appropriate amount

Water: Excessive

Water

Good operability is
obtained because of the
bearing effect.

PowderHydration reaction withwater produces hydrated
crystals on the particle
surface.

The hydrated crystals
become dense and thefinal strength increases.

CementWater

Cement crystals

SilicaThe spacing between theparticles is large, and theoverlap of the cement hydrated
crystals is weakened, thereby
decreasing the final strength.

Zirconia

Figure 3-13 Schematic diagram of Mielle setting with appropriate and excessive water12

3-4 Bismuth-free
General MTA cement contains Bismuth oxide as an X-ray contrast agent; however, the bismuth oxide
sometimes turns black, due to exposure to light. On the other hand, Mielle contains zirconia, which
has been successfully used as an X-ray contrast agent in various biological applications, such as indental materials and joint prosthesis. Zirconia is a material which is chemically stable and is notprone to discoloring. Therefore, color change of Mielle is unlikely to happen. The effect of different
X-ray contrast agents on biological safety is explained in the following sections.

3-5 Effect of different X-ray contrast agents on cells
As described above, in typical MTA cement compositions, oxides such as bismuth oxide and zirconia
are added to Portland cement as an X-ray contrast agent 5). This section presents a review of studies
examining the effects of each of the oxide powders used as an X-ray contrast agent on cells 6).
Materials and Methods
As samples, bismuth oxide (Bi2O3) and zirconia oxide (ZrO2) powders were used. Murine macrophagelike cells (RAW264.7) and murine osteoblast-like cells (MC3T3-E1) were used for the cells. D-MEM
supplemented with 10% FBS was used as a culture medium. Cells were seeded at 5 × 104 cells/mL on
culture plates, then cultured in a carbon dioxide incubator (37°C, 5 % CO2) for one day.

・Cell activity test
Oxide powders whose mean particle size
was adjusted to about 1 µm were added
to the culture medium to 0.01, 0.1, 1.0
and 10.0 0 mM, and after culturing foran additional 3 days, the activity of thecells was evaluated using WST-8 7, 8)(Figure 3-14).

NO2

SO3N
SO3-

NO2

SO3-

N
N+

H
N

NO2

N

Dehydrogen

MeO

SO3-

N
N

N

NO2

MeO

WST-8

WST-8 formazan

Cellular metabolic activity (dehydrogenase) changes WST-8 to orange WST-8 formazan
NO2

SO3-

H
N

SO3-

Orange: light

N
N

NO2

N
MeO

WST-8 is reduced to orange WST8 formazan by dehydrogenases(NAD+, NAD(P)+ dehydrogenase)that metabolize in living cells. By
measuring the intensity of this orange
shade as absorbance, it is possible toassess the effect of the sample on themetabolic activity of the cell.

High toxicity: The metabolic activityof the cells decreases and the production of
WST-8 formazan decreases
NO2

N
SO3-

Orange: dark

H

SO3-

N
N

N

NO2

MeO

Low toxicity: The metabolic activity of thecells is maintained, and the production of
WST-8 formazan is highFigure 3-14 Principle WST-8 test

That is, the darker the orange color (the higher the absorbance), the less the sample has an effect on
the metabolic activity of the cell.
・Transmission electron microscopy (TEM) observation
Cells were seeded, and powders with an average particle size of approximately 1 μm were added.
The cells were cultured for 3 days, and then pre-fixed with 2.5% glutaraldehyde diluted with PBS.
Thereafter, those post-fixed with osmium tetraoxide were dehydrated with ethanol series, andembedded in epoxy resin, to produce an ultrathin section with a thickness of 90 nm. Ultrathin sections
were stained electronically with uranyl acetate and lead citrate, and the cells were observed by TEM.

13

Results
In the cellular activity test, when powder with an average particle size of approximately 1 μm wasadded to RAW264.7 cells, the absorbance indicative of metabolic activity decreased significantly toless than 10% in the group supplemented with 0.1 mM or more of Bi2O3 powder against RAW264.7
compared with the control group. On the other hand, when zirconia powder was added, a decrease ofabout 50% was observed in the 10 mM addition group, but no decrease in absorbance was observed in
the 1 mM or less addition group (Figure 3-15).

Absorbance (relative to control)

1.4

Bi2O3

ZrO2

1.2
1
0.8
0.6
0.4
0.2
0
0.01

0.1

1

10

Powder concentration (mM)

Figure 3-15 Cellular activity upon adding sample powder to RAW264.7 cells
Subsequently, when the sample powder was added to MC3T3-E1 cells, the group supplemented withbismuth oxide powder in an amount of 0.1 mM or more showed an absorbance of 20% or less comparedwith the control group, indicating a significant decrease in cellular activity. On the other hand, in thegroup supplemented with zirconia powder, no decrease in absorbance was observed even in the groupsupplemented with 10 mM (Figure 3-16).

Absorbance (relative to control)

1.4

Bi2O3

ZrO2

1.2
1
0.8
0.6
0.4
0.2
0
0.01

0.1

1

10

Powder concentration (mM)

Figure 3-16 Cellular activity upon adding the sample powder to MC3T3-E1 cells
Figure 3-17 shows TEM images of each oxide powder added to RAW264.7 cells. Particles of each oxide
powder incorporated into the cytoplasm of RAW264.7 were observed. No obvious differences in cellmorphology were observed in the group supplemented with zirconia powder compared with the control
group, and no disruption images of the cell membrane or nucleus were observed. On the other hand, inthe group supplemented with bismuth oxide powder, ruptured and swollen cells and organelles wereobserved, with obvious cell destruction.

14

10 μmControl

10 μm

10 μm
Bismuth oxide

Zirconia

Figure 3-17 Incorporation of each oxide powder by RAW246.7cells
In conclusion, the effects of bismuth oxide and zirconia, which are contained as X-ray contrast agentsin MTA cement, on macrophage-like cells and osteoblast-like cells were investigated. The use ofchemically stable zirconia as an X-ray contrast agent may lead to an improvement in the biologicalsafety of MTA cement because bismuth oxide is stronger than zirconia for all cells in reducing cell
activity.
Next, the effects of MTA cements containing bismuth oxide
or zirconia on osteoblast-like MC3T3-E1 cells (Figure 3-18)
were examined.

Materials and Methods
As test materials, TMR-MTA cement Mielle, containing about
30% by mass zirconia, TMR-MTA CEMENT (the conventional Figure 3-18 Murine osteoblast-like cells
MC3T3-E1 cells
product), containing about 20% by mass zirconia, and prototype
MTA (Bis20, Bis30) containing 20% or 30% by mass bismuth oxide, were prepared. The sample powders
were kneaded with distilled water at a moisture ratio of 20% by mass and set for 24 hours at 37°C and 100%
relative humidity. To each test piece, 1 mL cell culture medium (MEMα with 10% FBS) was added per 1
cm2 of surface area, and the cell culture medium was soaked in the dark at 37°C for 24 hours. This immersion
liquid was collected and filtered through a 0.22 μm filter to be used as a test liquid, which was subjected to acell proliferation test.
・Cell proliferation study
MC3T3-E1 cells were seeded in 96-well culture plates at 1000 cells/well and cultured for 24 hours (5%CO2, 37°C). It was confirmed that cells adhered to the bottom surface of the wells, and the medium was
replaced with each test solution, and the cells in the wells were further cultured for 48 hours. Adding
10 μL of Cell Counting Kit-8 solution (Dojindo Laboratories) to each well and coloring for 2 hours todetermine the absorbance at 450 nm 7, 8).
Results and Discussion
Observation of the test specimens after immersion in the cell-culture medium revealed no change in
the appearance of the zirconia-containing Mielle and the conventional product, but black discoloration
was observed in Bis20 and Bis30, the bismuth oxide-containing products (Figure 3-19). Since the cell
culture medium used as the immersion medium contained components such as sugars and aminoacids, it was inferred that the blackening observed in Bis20 and Bis30 was caused by the reactions
between bismuth oxide and one or some of the components in the cell culture medium.

15

The cell proliferation test found that absorbance was less in the order Mielle to the conventional
product to Bis20 to Bis30 (Figure 3-20). Higher absorbance was observed for zirconia-containing
MTA cement versus bismuth-oxide-containing MTA cement. Although it has been reported that
MTA cement exhibits antimicrobial properties due to the strong alkalinity that occurs during thesetting process, the pH of the sample solutions was equivalent, and therefore, it was inferred that thedifference in eluted components affected the cell proliferation.

Before discoloration

0.8

After discoloration

Absorbance (450 nm)

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

Figure 3-19 Test specimens after immersion
in cell culture medium(Conventional, Mielle, Bis20, Bis30 from left)

Conventional
product

Mielle

Bis20

Bis30

Figure 3-20 Cell proliferation in each test solution

3-6 X-ray contrast
By increasing the ratio of zirconia, an X-ray contrast agent component contained in the composition,
the X-ray contrast has been greatly improved compared to the conventional product. The X-ray
contrast of dentine is said to be comparable to that of aluminum 1 mm thick, so the X-ray contrast ofdental materials is generally compared with that of aluminum based on JIS standard testing. In thissection, X-ray contrast of Mielle was compared with the conventional product.
Materials and Methods
Conventional and Mielle powder samples were kneaded with distilled water to 20 mass% moisture, anda 12 mm diameter and 1 mm thickness pellet-type sample was prepared using stainless steel braces.
X-ray imaging was then performed of a stepped wedge of aluminum with a stair-like structure of 1, 2,
3, 4,... mm in thickness (Figure 3-21), and the optical density of the X-ray contrast was calculated bycomparing the optical density of the X-ray image of the sample with the optical density of the image ofthe aluminum step of each thickness (Figure 3-22).
1mm

2mm

3mm

4mm

5mm

6mm

7mm

8mm

Aluminum step wedge

Figure 3-21 Schematic diagram ofaluminum step wedge

Conventional
product

MielleFigure 3-22 X-ray image schematic

16

Results and Discussion
The conventional product has an X-ray contrast of 3.4 mm in aluminum thickness, while Mielle has an
X-ray contrast of 5.4 mm in aluminum thickness, an increase of about 1.6 times (Figure 3-23).

X-ray contrast (mm Al)

The X-ray photograph taken by dental X-ray apparatus (Arm-type X-ray CT diagnostic apparatus
AUGE: Asahi Roentgen Ind. Co., Ltd.) is shown below (Figure 3-24). As is clear from the figure, thevisibility of the Mielle is greatly improved over the conventional product.

54

60 %UP

3
2

Conventional
product

1

MielleBismuth oxide
prototype0

Conventional product

MielleFigure 3-23 X-ray Contrast

Figure 3-24 X-ray photographs of 1-mm-thickpellets of MTA cement

In actual clinical practice, the affected area may be small and the amount of MTA cement used may bevery small. Therefore, in order to confirm the visibility of MTA cement on X-rays when applying small
amounts in patches in the clinic, a dental model (Figure 3-25) was filled with the conventional productand Mielle and an X-ray was performed.

Figure 3-25 Dental model filled with MTA cement(Left: Conventional product, Right: Mielle)

Figure 3-26 Confirmation by X-ray photography(Left: Conventional product, Right: Mielle)

Even where very small amounts of MTA cement Mielle are used, it is possible to see the MTA filled area
more easily than conventional product. (Figure 3-26).

3-7 Strong alkalinity and sustained release of calcium ions
As described in the previous section, the X-ray contrast of Mielle has been improved by increasing theratio of zirconia compared with the conventional product.
In this section, the following experiments were conducted to verify the strong alkaline and calcium
sustained release levels and compare them with the conventional product.
Materials and Methods
The conventional product and the powder sample of Mielle were kneaded with distilled water at amoisture ratio of 20% by mass to prepare a pellet-type sample (for pH test) having a diameter of 6mm
and a thickness of 1 mm, and a rectangular parallelepiped sample (for sustained calcium ion release17

test) having a length of 33 mm, a width of 13 mm and a thickness of 2 mm. Thereafter, the samples
were set in an environment of 37°C and 100% relative humidity for 24 hours, then immersed in 10 mLof distilled water, and the pH and the amount of calcium ion released in the immersion solution after
one day were measured.
Results and Discussion
Figure 3-28 shows the results of measuring the pH of immersion solutions obtained by separately
immersing pellets of the conventional product and Mielle in distilled water for one day using a pH
meter (F-55: Horiba, Ltd., Figure 3-27).
12

pH

11
10
9
8
7

Conventional productFigure 3-27 pH meters

MielleFigure 3-28 pH after 1 day in immersion solution

The immersion solution exhibited a high pH of 11.5 for both the conventional product and the Mielle
and an equivalent strong alkalinity.

Sustained release of calcium ions
(μg per cm2)

Next, Figure 3-29 shows the results of measuring the sustained release of calcium ions in the immersion
solution using an ICP-emission spectrometer (SPS3500DD: Hitachi High Technology Science Co.,
Ltd.) after separately immersing rectangular parallelepiped samples of the conventional product andMielle in distilled water for one day.

600
500
400
300
200
100
0

Conventional product

MielleFigure 3-29 Calcium ion sustained release
after 1 day in immersion solution
Calcium ion sustained release of the conventional-product and Mielle were comparable.
The weight ratio of zirconia to the conventional product was increased in Mielle, and this compositionchange was found to improve the X-ray contrast while not affecting the strong alkalinity and calcium
sustained release.

18

3-8 Enhancement of hard tissue formation
MTA cement has been reported to promote the formation of hard tissues in surrounding tissues whenit acts on the exposed pulp. In addition, hard tissue formation just under the pulp exposure has beenpreviously confirmed by direct pulp capping for rat molars 1). The details will be discussed later, but
the volume ratio of the cement component involved in hard tissue formation is the same as that of theconventional product. Accordingly, it is expected that Mielle has the same effect as the conventional
product in promoting hard tissue formation. Therefore, in this section, the following tests were
performed using Mielle (Figure 3-30).
Dental nerves exposed

Glass ionomer cement
MTA cement Mielle

Confirm formation of hard tissue

Nervous

Sealing of exposed pulp with MTA
cement Mielle and temporary sealing
with glass ionomer cement

Figure 3-30 Schematic representation of the rat pulp capping test

After pulpotomy in 8-week-old female rats, Mielle was patched on and temporarily sealed withglass ionomer cement. Two weeks later, the surrounding tissue including the patch site was fixed
in 4% paraformaldehyde-containing PBS for 24 hours. Sections were prepared by degreasing anddecalcification, followed by Hematoxylin-Eosin staining and observation by light microscopy.
As shown in Figure 3-31, 2 weeks after the patch application, the formation of hard tissue with
sufficient thickness to completely cover the exposed pulp surface was found under the treated lesion.
From the above, it has been clarified that Mielle has an effect of promoting the formation of a hardtissue of the surrounding tissue, similarly to the conventional product.

MTA patch site
Hard tissue

Figure 3-31 Hard tissue formation after direct pulp capping (rat molars, 2 weeks after application)
Image courtesy of Health Sciences University of Hokkaido

193-9 Enhancement of X-ray Contrast and Maintenance of the Strengths of the Conventional ProductThe increased zirconia ratio which distinguishes
Mielle from the conventional product is associatedwith a decrease in cement content (calcium silicate).
Calcium silicate is an ingredient directly linked tothe important properties of MTA cement i.e., setting,
being strongly alkaline, and exhibiting sustained
release of calcium ions. Therefore, the pH of theimmersion solution and the sustained release amountof calcium ions have been confirmed to be equivalent
to those of the conventional product, as shown in
Section 3-7. This section discusses the reasons why thephysical properties have not changed.

Spherical silica
particle

Zirconia particulatematterFigure 3-32 Appearance of each micro particle
at the same weight (5 g)

In Mielle, the zirconia ratio is increased by replacing some of the spherical silica fine particles which
are included mainly for the purpose of improving operability with zirconia fine particles.
Silica and zirconia have densities of about 2.2 g/cm3 and about 5.5 g/cm3, respectively, and so thevolume of silica is about 2.5 times larger than that of zirconia when the weights are the same (Figure3-32).
Due to this difference in density, the volume ratio of calcium silicate in Mielle was equivalent to thatof the conventional product (Figure 3-33). In the comparative verification of physical properties asshown in Sections 3-2, 3-3, and 3-7, reflecting these facts, Mielle and the conventional product wereconsidered to show equivalent physical properties.

Volume proportion ((vol%)Cement Components;

Zirconia Micro particles;

Spherical Silica Micro particles

75

75

Conventional product

MielleFigure 3-33 Volume ratio of ingredients of theconventional product and Mielle

3-10 Adhesion Strength with CR System
The adhesion strength with the CR system wasmeasured assuming composite restoration after
pulp protection by Mielle. The test sample wasprepared by using Mielle and TMR-AQUABOND
0 (YAMAKIN CO., LTD.) as adhesive material,
and TMR-Z Fill 10. (YAMAKIN CO., LTD.) ascomposite resin.
First, hardened Mielle was set into a silicon mold,and a smooth surface was formed using waterresistant abrasive paper.

20

4. Resin cement
3. TMR-Z Fill 10.
2. TMR-AQUA BOND 0

1. Hardened Mielle

Figure 3-34 Structural Schematic Diagram of
Adhesive Test Specimens

The surface was masked with tape containing holes with a diameter of 5 mm to limit the adhesive
surface. Next, TMR-AQUABOND 0 was applied to the adhesive surface of Mielle and processed as
usual.
TMR-Z Fill 10. was then filled and photopolymerized using LED light irradiator, and a stainless steel
rod was attached using resin cement to make the test body. Samples were stored in water at 37℃ for 1
day and 7 days. After that, a test was carried out using a small universal tester (EZ-Graph: Shimadzu
Corporation) to pull the stainless steel rod perpendicularly to the bonding surface at a rate of 1 mm
per minute, and the stress at break was defined as the tensile adhesion strength.(Figure 3-34).
The results of the adhesion test are shown in Figure 3-35.

Tensile bonding strength（MPa）

6

In water at 37°C

4

2

0

After 1 days

After 7 days

Figure 3-35 Tensile adhesive strength between Mielle and TMR-AQUABOND 0
As a result of this study, it was confirmed that TMR-AQUABOND 0 showed constant adhesive strength
between MTA cement and CR. This study was limited to adhesion between MTA cement surfaces of
5 mm diameter and CRs, but in the clinic, the surfaces of teeth other than MTA cement in the cavity
also become bonding surfaces, so it is considered that more firm adhesion can be obtained.
TMR-AQUABOND 0 uses M-TEG-P (Figure 3-36), which is an amphiphilic monomer.
M-TEG-P® is a registered trademark of YAMAKIN CO., LTD.

O
O

O

O

O

O

Polymerizing group

Tetraethyleneglycol part

O

P

OH

OH

Phosphate group

Characteristic properties- Demineralization effect by phosphate group can be controlled by organic group- Higher detergency than phosphoric acid
- Improvement of compatibility

Figure 3-36 Chemical structures and properties of M-TEG-P
The use of M-TEG-P allows TMR-AQUA BOND 0 to exhibit hydrophilicity, so it is consideredthat it has excellent compatibility with water-containing Mielle surfaces and adheres closely to theunevenness of cement surfaces (Figure 3-37). It is also known that phosphoric monomers adhere well
to zirconia 9-11), and it is thought that the zirconia contained in Mielle and M-TEG-P phosphate groupbind to each other for effective adhesion (Figure 3-38).

21

M-TEG-P

P

EG-

M-TEG-P

M-T

EG

-P
TEG
M-

-T
M
-P

TMR-AQUA BOND 0
TMR-MTA cement Mielle

TMR-MTA cement Mielle

Figure 3-37 Image of interface

Zirconia Fine Particles

Figure 3-38 Binding between M-TEG-P and Zirconia

After treatment of pulp protection by excellent setting property TMR-MTA cement Mielle, TMRAQUA BOND 0, which provide stable adhesion even in moist conditions can be used. Moreover, there
is direct composite TMR-Z Fill 10. which can expect strengthening of tooth and preventing bacteria
to stay on tooth by releasing fluoride. These products achieve a complete system from pulp capping tofilling as TMR Series.

CR
BOND
MTA

MTA

＋

BOND

＋

CR

＝

TMR Series

Figure 3-39 Innovation through the TMR Series
References
1) Kobayashi, K.: “The Latest Concrete Technology (Saishin konkurito kougaku)”, 5th ed.: MORIKITA PUBLISHING CO., LTD., Tokyo, 2010.
2) Takahiro Kato, Ritaro Matsuura, Teruo Anraku, Sayaka Sakakibara, Kazuhiko Endo: “Development of New Calcium Silicate Cement (Shinki keisan
karusiumukei semento no kaihatu) ” – Effect of moisture content on curing time of cement- The 144th Meeting of the Japanese Society of Conservative
Dentistry, p.114, 2016.
3) Fridland M, Rosado R: Mineral trioxide aggregate solubility and porosity with different water-to-powder ratios. J Endod, 29, 814-817. 2003.
4) Takafumi Nakano, Takahiro Kato, Ritaro Matsuura, Teruo Anraku, Sayaka Sakakibara, Kazuhiko Endo: “Development of New Calcium Silicate
Cement”(Shinki keisan karusiumukei semento no kaihatu)” 2nd Report – Effect of moisture ratio on compressive strength - The 145th Meeting of theJapanese Society of Conservative Dentistry, p.129, 2016.
5) Chihiro Kobayashi : Clinical Application of MTA- Aiming for better endodontic healing (MTA CEMENT no rinshou yori yoi endo no Tiyu wo mezashite)
Ishiyaku Publishers, Inc., Tokyo, 2013.
6) Hirokazu Toshima: Cellular Response to Oxidized Powder contained in MTA cement(MTAsemento ni haigou sareteiru sannkabutu funnmatu ni taisuru
saibou no hannou) Doctoral Dissertations, 2015.
7）Ishiyama M, Miyazono Y, Sasamoto K, Ohkura Y, Ueno K: A Highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as
well as cell viability. Talanta, 44(7), 1299-1305, 1997.
8) Tominaga H, Ishiyama M, Ohseto F, Sasamoto K, Hamamoto T, Suzuki K, Watanabe M: A water-soluble tetrazolium salt useful for colorimetric cell
viability assay. Anal Commun, 36, 47-50, 1999.
9) Shimoe S, Hirata I, Otaku M, Matsumura H, Kato K, Satoda T: Formation of chemical bonds on zirconia surfaces with acidic functional monomers.
Journal of Oral Science, 60(2), 187-193, 2018.
10) SS Atsu, MA Kilicarslan, HC Kucukesmen, PS Aka: Effect of zirconium-oxide ceramic surface treatments on the bond strength to adhesive resin.
Journal of Prosthetic Dentistry, 95(6), 430-436, 2006.
11) A Piwowarczyk, H-C Lauer, JA Sorensen: The shear bond strength between luting cements and zirconia ceramics after two pre-treatments. Operative
Dentistry, 30(3), 382-388, 2005.

22

4

MTA cement biological safety evaluation
Table 4-1 presents the biological safety considerations for medical devices for dental use. Medical
devices for dental use in contact with the human body should be classified according to contact site(non-contact devices , surface contact devices , internal and external devices, implanted devices) andduration of contact (temporary contact, short-and medium-term contact, long-term (permanent)
contact) and evaluated for biological safety as appropriate. “Evaluation of biological safety” refers
to the risk assessment of factors (e.g., cytotoxicity, sensitization, genotoxicity) that may adversely
affect human health, and sale of medical devices for dental use is permitted after such assessment of
biological safety.
Section 3-5 demonstrated that zirconia (ZrO2) has less effect on cells than bismuth oxide (Bi2O3)
as an X-ray contrast agent. This section presents examples of biological safety evaluation studies
of cytotoxicity, sensitization, and intradermal reactions that require risk assessment for all medical
devices for dental use in contact with the body to assess the biosafety of MTA cement using zirconia asan X-ray contrast agent.
Table 4-1 Biological safety considerations in medical devices for dental use
Category ofMedical devicefor dental use

Contact period

Biological tests

○

C

○

○

○

A

○

○

○

B

○

○

○

C

○

○

○

A

○

○

○

○

○

○

○

Implantation○○

Genotoxicity○○

C: Long-term (permanent)(Beyond 30 days)

Subacute (or subchronic)
systemic toxicity

Skin irritation and intradermal
reactions
Delayed-type hypersensitivity(sensitization)○

B

B: Short-and medium-term(24 hours to 30 days)

Acute systemic toxicity

Cytotoxicity
A

A: Temporary(Within 24 hours)

Non-contact devices
Skin

Surface contact
devices

Oral tissue (mucosa)

Damaged surface
Device thatconnects the bodywith the outside
Implanted device(Tissue/bone/teeth)(Tissue/bone)

B

○

○

○

C

○

○

○

A

○

○

○

B

○

○

○

○

○

○

○

C

○

○

○

○

○

○

○

A

○

○

○

B

○

○

○

○

○

○

○

C

○

○

○

○

○

○

○

4-1 Cytotoxicity test 1)“Cytotoxicity” refers to the ability of cells to inhibit functions such as proliferation or metabolic
inhibition, or to cause cell death. Cytotoxicity has the potential to lead to toxicity to tissues, organs,and thus organisms. Therefore, evaluation of cytotoxicity is required for medical devices for dental use
in any contact, even minor contact, with the human body.
Various methods exist for the evaluation of cytotoxicity, but here we performed a cytotoxicity study

23

using V79 cells (Chinese hamster lung-derived fibroblasts) according to ISO 10993-5: 2009, Biologicalevaluation of medical devices-Part 5: Tests for in vitro cytotoxicity.
Extracts were prepared by immersing MTA cement in cell culture medium MO5.
After culturing the V79 cells in this extraction solution, cell colonies (cell groups composed of 50 ormore cells) that are formed were counted. As shown in Figure 4-1, even the 100% concentration showeda high colonization rate of approximately 80%. When the colonization rate of the extract at 100% is
70% or more, it is assessed as not cytotoxic.
120

Colony efficiency (%)

100
80
60
40
20
0

0

20

40

60

80

100

Concentration of test solution (%)

Figure 4-1 Colonizing V79 Cells in MTA cement

4-2 Sensitization test 2)
Some chemicals cause allergic inflammation when in contact with biological tissues. As an allergy
related to the dental material, the delayed type allergy represented by the metal allergy can be
mentioned. Sensitization tests were conducted using guinea pigs in accordance with ISO 10993-10:
2010, Biological evaluation of medical devices-Part 10: Tests for irritation and skin sensitization, a test
to evaluate the delayed allergenicity of dental materials.
First, a test solution is prepared by extraction with physiological saline from MTA cement at 121°C
for 1 hour. The test solution and Freund's complete adjuvant, a sensitization potentiator, are injected
intradermally into guinea pigs to induce primary sensitization, and the test solution is applied (openor closed) to the guinea pig intradermal injection site to induce secondary sensitization. Following theinduction of secondary sensitization, the test solution is used to induce sensitization and observe andnumerically evaluate the skin changes (erythema and oedema) for the sensitization potential of the test
material (Table 4-2).
Table 4-2 Skin changes
Findings

Score

No apparent response0

Scattered or patchy erythematous plaques

1

Moderate erythema covering the entire
application site

2

Marked erythema and edema

3

When the test solution was applied closed, no skin reactions were observed at any of the applicationsites (all scores: 0). Based on the above, the saline extract of MTA cement was not considered to besensitizing under the conditions of this study.

24

4-3 Intradermal response testing 2)
The irritation test (intradermal response test) is a test to evaluate the injurious, pro-inflammatory, andirritant properties of medical devices for dental use in biological tissues. Appropriate test methods
should be selected and evaluated based on the site of medical devices for dental use contact, including
intradermal test (contact with tissues in vivo), skin irritation test (contact with skin), and eye irritation
test (contact with eyes). This section presents an intradermal test in rabbits.
Intradermal response tests were performed according to ISO 10993-10:2010, Biological evaluation of
medical devices-Part 10: Tests for irritation and skin sensitization.
Extract solution (test solution) was obtained from MTA cement by extraction with physiological salineor sesame oil at 121°C for 1 hour. This was administered intradermally to the test animals (rabbits) as
the test solution. Skin reactions were observed immediately after administration and 24, 48, and 72
hours after administration to verify the formation of erythema and edema at the administration site of
the test solution.
No skin reaction was observed in the saline extract at any observation time. On the other hand, mild
erythema was observed in the sesame oil extract, but similar erythema was observed in blank extract,
and the differences between the extract and blank extract were 0. Based on the above, MTA cement wasconsidered to have acceptable risks for intradermal reactions.
This paper presents biological safety assessment of MTA cement in actual use, as required by thelaw and legal system (the Pharmaceutical and Medical Device Act). In addition to the biologicalsafety tests introduced in this study, only materials that have been evaluated for biological safety inconsideration of the manner of use of the materials and confirmed to have no potential problem in anyof the evaluation items are actually approved as products.

References
1）ISO 10993-5:2009, Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity2）ISO 10993-10:2010, Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization25

5

Discoloration of MTA cement by Exposure to Light
MTA cement has excellent functions such as antimicrobial action, sealing ability, biocompatibility, andhard tissue inducibility and is used in applications such as direct pulp capping. X-ray contrast agents
have been added to MTA cement for prognostic follow-up. Various types of X-ray contrast agents havebeen used in different products, including bismuth oxide, zirconia, and tantalum oxide. Because theworld’s first MTA cement used bismuth oxide, bismuth oxide has been added in many MTA cement
products.
Although MTA cement has various functionalities as described above, problems of discoloration by
blackening over time have been reported in recent years 1). The cause of this is that bismuth oxidechanges to black due to a reduction reaction (oxygen-deficient state) due to adhesion of blood whenhemostasis is insufficient in treatment, reaction with sulfur in proteins, exposure to ultraviolet rays
under oxygen blocking conditions, and the like.
Instead of bismuth oxide, in Mielle, zirconia is added as an X-ray contrast agent to prevent blackening
due to this exposure.
In this section, we examine the blackening of MTA cement caused by exposure to ultraviolet light.
Examination of such blackening was undertaken for X-ray contrast agents (zirconia and bismuthoxide) as simple substances , the prototype which contains bismuth oxide, and MTA cement Mielle,
containing zirconia instead of bismuth oxide. In addition, the biological safety effects of thisblackening 2) are also verified. XXX

5-1 Discoloration verification of zirconia and bismuth oxide
Materials and Methods
Zirconia powder and bismuth oxide powder were filled as simple substances into a mold made ofsilicon. Each powder was photoirradiated using an ultraviolet lamp (UV light: Hi-STRON N-900L,
Kyowa Irika Co., Ltd.) with and without application of glycerin, and the color tone was evaluatedvisually.
Results and Discussion
Figure 5-1 shows photographs of zirconia
powder and bismuth oxide powder
as simple substances before and after
exposure to light with ultraviolet lamp.
Zirconia was not altered by exposure tolight. On the other hand, bismuth oxide
showed no color change due to exposure
without glycerin coating, but blackening
was observed with glycerin coating. It is
presumed that the bismuth oxide became
oxygen-deficient by reduction reaction
by being exposed under conditions inwhich oxygen was blocked by the glycerin
coating, and blackening occurred.

26

UV light
Test sample

Without
glycerincoating

IRRADIATION TIME
0 min

15 minutes

30 minutes

Zirconia

Bismuth oxide

Withglycerincoating

Zirconia

Bismuth oxide

Figure 5-1 Color changes induced by ultraviolet
irradiation of zirconia powder and bismuth oxide
powder as simple substances5-2 Effects of X-ray Contrast Agent on Color Change in MTA cement Composition
Materials and Methods
Mielle mixed with 30% by mass of zirconia and a prototype MTA cement mixed with 20% by massof bismuth oxide was kneaded at a moisture ratio of 20% by mass and filled into a silicone mold.
Glycerin was applied immediately after filling, and changes in color tone were assessed visually after
photoirradiation with LED light irradiators (LED light: G-Light Prima II, GC Corporation).
Results and Discussion
Figure 5-2 shows the change in appearance over time of Mielle, containing zirconia, coated withglycerin, and the prototype MTA cement, containing bismuth oxide, coated with glycerin, whenilluminated by an LED-illuminator. No color change due to light irradiation was observed in Mielle.
On the other hand, blackening was observed in the prototype MTA cement, and the discoloration
tended to be stronger as the duration of irradiation increased.
Exposure time with LED light irradiator
0 seconds

10 seconds

30 seconds

60 seconds

60 seconds

Mielle(White)Mielle(Light Ivory)
Prototype productcontaining
20% bismuth oxide

Figure 5-2 Light exposure test with LED light irradiator of Mielleand prototype containing bismuth oxide

5-3 Effect of blackening on biosafety
As described above, the zirconia-containing MTA cement does not undergo discoloration due to light
exposure, while the bismuth oxide-containing MTA cement does . It has also been shown in Section 3-5
that fine powdered bismuth oxide with an average particle size of 1 μm is taken up by cells and causes
damage to cells. If the blackening is caused by the conversion of bismuth oxide to a more chemicallyunstable state, the blackened MTA cement may have some effect on the biological safety. Therefore,
the effects of blackening in bismuth oxide-containing MTA cements on 1) the elution of components
and 2) the cytotoxicity to human monocytic leukemic cells and human mucosal epithelial cells areinvestigated in this section 2).
Materials and Methods
MTA cement (hereinafter referred to as “Zr-n”) containing 20% by mass of zirconia and a prototype
of MTA cement (hereinafter referred to as “Bi-n”) containing 20% by mass of bismuth oxide
were kneaded at a moisture ratio of 20% by mass, filled in a mold made of silicone, and set for
24 hours. After coating the surface of Bi-n sample with glycerin, the surface was irradiated witha photopolymerization device (LED CURE Master, marketed by YAMAKIN CO., LTD.) for 180
seconds to prepare a blackened sample (hereinafter referred to as “Bi-black”) (Figure 5-3). It has been
confirmed that Zr-n does not blacken with the same treatment.

27

Exposure

Zr-n

Bi-n

Bi-black

Figure 5-3 Prototype MTA cement containing bismuth oxide(before and after light exposure) and Zr-n1) Elution test
The samples were immersed in a mixed solution of 0.1 M lactic acid and 0.1 M sodium chloride (1 mL/cm2 ) and allowed to stand at 37°C for 7 days. The eluted components in the immersion liquid were
measured using an ICP-emission spectrometer (SPS3500DD, Hitachi High-Tech Science Corporation).
2) Trypan blue dye exclusion test 3)
A sample of 15 mm in diameter and 1 mm in thickness was placed in the wells of 24-well culture plates,
where a human monocytic leukemia cell line (THP.1 cell) was seeded (10.0×104 cells/well). After 72
hours of incubation in a carbon dioxide incubator, the cells were mixed with trypan blue, and living
and dead cells were counted separately on a hemocytometer (Figure 5-4).

Trypan blue

Living cell

Living cell

Dead cell
Trypan blue

Dead cell

Figure 5-4 Principle of trypan blue dye exclusion test3) WST cell proliferation test 4, 5)
Samples were immersed (1 mL/3 cm2) in serum-free culture medium for keratinocytes for 72 h. Wellsof 96-well culture plates were seeded (1.0×104 cells/well) with human mucosal epithelial cells (RT-7
cells) and cultured in a carbon dioxide incubator for 24 h. The culture medium of the cell was changed
to immersion solution and cultured for another 48 hours. 10 μL of Cell Counting Kit-8 (Dojindo
Chemical Laboratories, Inc.) agent was added. The cells were then colored for 2 hours, and absorbanceat 450 nm was measured.
Results
1) Elution test
The elution of Mg, Sr, Al, Si, Ba, B, K, and Ca was observed from all samples. Among these eluates,
the elution of Ca was remarkable at >1200 ppm in all samples. There was no elution of Zr from
Zr-n for the elution derived from the X-ray contrast medium contained in the samples. On the other
hand, Bi was eluted from Bi-n and Bi-black. In addition, the amount of Bi eluted was in the order Biblack> Bi-n, confirming the increase of Bi elution by blackening in MTA cements containing bismuthoxide(Figure 5-5). It was presumed that this was because bismuth oxide caused oxygen deficiency due
to light exposure under oxygen deprivation, and bismuth was generated.

28

( ＞1200 ppm)
25

Zr-n

Bi-n

Bi-black

Bi

Mg

Sr

Elution (ppm)

20
15
10
5
0

n.d.

Al

Si

Ba

B

K

Ca

Figure 5-5 Eluted components of each MTA cement
2) Trypan blue dye exclusion test
A prototype MTA cement containing silica in Portland Cement was used as a control sample(PC).
When THP.1 cells were cultured on the samples and subjected to the trypan blue dye exclusion test,
the increase in cell number was in the order PC ≧ Zr-n > Bi-n ≧ Bi-black (Figure 5-6). Cell numbers
were greatly increased on PC and Zr-n from the number of seeded cells (10×104 cells/mL), while only

Cell number (×104 cells/mL)a slight increase was observed in Bi-n and Bi-black, with no significant difference between Bi-n and Biblack. In addition, cell viability was more than 90% in all samples, and there was no cytotoxicity that
resulted in cell death (Figure 5-7).

80

60

40

20

0

PC

Zr-n

Bi-n

Bi-black

Figure 5-6 Growth of THP.1 cells cultured on MTA cement

Cell viability (%)

100.0
80.0
60.0
40.0
20.0
0.0

PC

Zr-n

Bi-n

Bi-black

Figure 5-7 Cellular viability of THP.1 cells cultured on MTA cement293) WST cell proliferation test
The absorbances based on the metabolic activity of the cells were in the order Zr-n > PC > Bi-black≧ Bi-n (Figure 5-8). Similar to the trypan blue dye exclusion test, no significant differences were foundbetween Bi-n and Bi-black in this method.

Absorbance (450 nm)

1.600

1.200

0.800

0.400

0.000

PC

Zr-n

Bi-n

Bi-black

Figure 5-8 Metabolic activity of RT-7 cells cultured on MTA cement
In conclusion, the elution test for the X-ray contrast material used for each cement sample showedthat zirconia was not eluted from Zr-n containing zirconia, but Bi was eluted from Bi-n and Bi-blackcontaining bismuth oxide. Cell testing showed good cell proliferation in zirconia-containing cementcompared to bismuth oxide-containing cement.
An increase in the amount of Bi eluted was observed in bismuth oxide-containing cements when
blackening was produced by exposure under oxygen deprivation. On the other hand, there were no
significant differences between Bi-n and Bi-black in any of the cellular tests, and no effect of bismuthoxide on cytotoxicity was observed.

References
1) Fridland M, Rosado R: Mineral trioxide aggregate (MTA) solubility and porosity with different water-to-powder ratios. J Endod, 29, 814-817, 2003.
2) Ritaro Matsuura, Takahiro Kato, Kazuhiko Endo, and Tetsuya Yamamoto: The effect of blackening of MTA cement on biological safety. Lecture Abstracts,
p113, 2017, Autumn Academic Congress (147th) of the Japanese Society of Conservative Dentistry.
3) Correa GT, Veranio GA, Silva LE, Hirata Junior R, Coil JM, Scelza MF: Cytotoxicity evaluation of two root canal sealers and a commercial calcium
hydroxide paste on THP1 cell line by Trypan Blue assay.J Appl Oral Sci, 17(5), 457-461, 2009.
4) Ishiyama M, Miyazono Y, Sasamoto K, Ohkura Y, Ueno K: A Highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as
well as cell viability. Talanta, 44(7), 1299-1305, 1997.
5) Tominaga H, Ishiyama M, Ohseto F, Sasamoto K, Hamamoto T, Suzuki K, Watanabe M: A water-soluble tetrazolium salt useful for colorimetric cell
viability assay. Anal Commun, 36, 47-50, 1999.

30

6

How to operate TMR-MTA cement Mielle

6-1 Operation Procedure
Adjustment method
The kneading (mixing) of the powder and liquid is adjusted according to the case, based on thestandard moisture ratio. Since the material comes into direct contact with nerves, the equipment usedshould be sterilized and purified water should be used. Caution should be exercised when using metal
instruments, as strong forces can cause the metals to shave and darken. The procedure is shown in
Figure 6-1.
1. Indications

2. Cleaning

・Uninfected pulp is indicated if cavity formation or trauma
accidentally leads to exposure within 2 mm of the pulp.
※ Infected pulp is not indicated.
※ During cavity formation, treat with sterile steel bars or the like.

1. Clean the exposed pulp surface with sterile saline or the like. When
disinfecting the exposed surface, disinfect with 3 to 10% aqueous
sodium hypochlorite before washing.
2. Dry with sterile cotton balls and check for hemostasis.
※ If hemostasis is difficult, intense inflammation may be present in the pulp, andpulpotomy or pulpectomy is indicated.

Caries-affected area

Exposed pulp

Dentalpulp

Dentalpulp

Removal of caries-affected areas

Cleaning

Confirmation of drying and hemostasis

3. Pulp capping by TMR-MTA cement Mielle
1

2

3

4

Collect powder on mixing paper or
glass plate.

Purified water is dropped near the
powder. (Standard powder ratio: 0.2 g
powder/water 0.05 g)

The powder and purified water are
kneaded using a spatula for about 30
seconds to form a uniform paste.
※ Adjust the viscosity of the paste byadding purified water as necessary.

Using a carrier tool, etc., cover the
exposed pulp with a scouring powder.
(Operating time: about 3 minutes after
scouring)※ For 3 g container: Measure the powder with the attached spoon to
make two cups (about 0.12 g). One drop of purified water (about 0.03 g)from the attached ophthalmic solution container is equivalent to theapproximate standard ratio of thepowder (moisture ratio: 20%).
×2

MTA cement

MTA cement Mielle exhibits poor setting when fluid is excessive. Be sure to remove excess water with sterilecotton balls when washing with water after applying MTA cement Mielle.

Figure 6-1 How to Use Mielle
Movie

How to use TMR-MTA cement Mielle
and Moisture Ratio
Summary for mixing procedure.

Pulp Capping method
Since this product can be kneaded with a small amount of water and sets quickly, it is possible totemporarily seal the filled portion with glass ionomer cement immediately after filling the exposed
pulp portion with paste and removing excess paste and moisture with a cotton ball or the like.

31

4. Temporary sealing

5. Final restoration

1. After pulp capping the exposure site with
MTA cement, remove excess MTA
cement and water with a sterile cottonswab.
2. Temporary seal by glass ionomer cement.
It can be temporarily sealed immediately
after pulp capping.

Glass ionomer cement

1. After follow-up, remove some of the glassionomer cement to the extent that MTA
cement does not peel off.
2. After bonding, a final restoration procedure
is performed with composite resin.

Composite resin
Glass ionomer
Cement※Filling and crown formation after direct pulp capping
should be done after at least one month of follow-up.

Figure 6-2 Flow of final restoration from temporary sealing

1. Preoperative

2. Removal of softened dentine(pseudo-exposed) pulp

5. Preoperative dental

3. Application of MTA

4. Temporary sealing

6. Postoperative dental

Figure 6-3 Clinical case of direct capping 1* Images are from a patient whose tooth was temporarily sealed with glass ionomer cement
after pulp capping with TMR-MTA cement Mielle.
Photographs courtesy of Professor Masato Saito, Health Sciences University of Hokkaido

Figure 6-4 Clinical case of direct pulp capping 2* Images are from a patient whose tooth pulp was capped with TMR-MTA cement Mielle, and after that
gutta-percha covering the upper part of the MTA, and bonding followed by restoration with composite resin.
Image courtesy of Professor Susumu Sudo, Nankodai Dental Clinic, Izumi-ku, Sendai City32

In addition, the Mielle lineup consists of two colors (white and light ivory) for each color tone of theglass ionomer cement used for temporary sealing, and we recommend that it be used separately, withwhite for the crown color if the glass ionomer cement is a color such as A3, and light ivory if it is white.

6-2 Operating time and initial setting time by moisture percentage
The typical moisture ratio of the Mielle is 20 mass%, but if the paste is hard to operate or is dried anddifficult to operate, it is easy to adjust the viscosity of the paste by adding water prior to starting thesetting. It is also possible to adjust the viscosity and duration of operation of the paste by increasing
the moisture ratio to 25-30 mass% as shown in Table 6-1.
Table 6-1 Operating time and initial setting time by moisture ratio
Moisture ratio (powder : water)

Paste property

Operating time

Initial setting time

20 mass% (powder 0.2 g : water 0.05 g)

Standard

About 3 minutes

15-30 min

25 mass% (powder 0.2 g : water 0.07 g)

Soft

About 6 minutes

30-40 min

30 mass% (powder 0.2 g : water 0.09 g)

Very soft

About 9 minutes

40-60 minHowever, since the initial setting time tends to be longer when the paste is filled in a soft state, as
shown in Table 6-2, it is possible to shorten the initial setting time by removing excessive moisture witha dry cotton swab or the like after the paste is filled.
Table 6-2 Moisture percentage and initial setting
time with and without wiping
Moisture ratio

No wipingWith wiping

20 mass%

15 minutes

5 minutes

25 mass%

30 minutes

5 minutes

30 mass%

60 minutes

10 minutes

33

Special
FeaturePoints of precision therapy with MTA

1Rubber dam moisture protection to prevent pulp infection

It is imperative to use rubber dams in pulp preservation treatments. When removing
restorations, a rubber dam is placed in advance to prevent accidental ingestion and aspirationof dislodged restorations, and it also facilitates transfer to direct pulp capping in the event of
exposure of the pulp.
When using a variety of chemical solutions, a rubber dam can be used for safety.

2

Thorough sterilization of equipment

Complete cleaning, disinfection, and sterilization of the instruments used are required in pulppreservation treatment. When using MTA cement, spatulas, kneading plates, filling tools, etc.
must be washed and sterilized.

Wash Out Evaluation

3

MTA cement selection

MTA cement often needs to be used unexpectedly for direct pulp coverage.

Color Stability

For this reason, the speed of setting is important in clinical practice.
Unset cement may be discharged by contact with tissue fluid (washout) and it may not achieve
full performance.
In addition, considering aesthetics, it is important to select products that are not prone todiscoloration after pulp capping.

4

Use of a microscope to increase treatment accuracy

The use of a microscope ensures a bright surgical field that has been enlarged tens of times. This
will ensure removal of infected dentine and minimize invasion, thereby significantly reducing
patient burden. Microscopes can also record the treatment content in photographs and videos,
allowing patients access to a detailed description of the condition of the affected teeth, which
increases patient understanding and satisfaction.

5

Diagnosis by Dental Cone Beam CT

Three-dimensional images of CT can inform the dentist of the structure and the shape of theteeth and what problems are occurring in the teeth. Dental x-rays alone provide only twodimensional planar information, so treatment must rely on the practitioner’s experience andintuition in many areas, whereas CT can anatomically identify the internal condition of the
tooth, allowing for proper treatment planning and shortening of the treatment time.

Although MTA cement is a material with good sealability and excellent performance, it is important to use
rubber dams and microscopes and to use it correctly, while take appropriate measures against infection. In
other words, it should be noted that the use of MTA cement does not increase the rate of successful pulp
preservation treatment. For more information on using MTA cement , please refer to Shinai iryoho nosanshu no shinki (“The Three Sacred Treasures of Endodontic Therapy”; Dental Diamond Co.) pp.138-141.

34

Mitsuhiko
Takata, D.D.S.
Takata Dental Clinic
Chuo-ku, Kobe

7

Conclusion
Mielle is an MTA cement with superior usability and biocompatibility. Like the conventional product,
it is bismuth-free, and the X-ray contrast has been enhanced by 60% over the conventional product.
Therefore, since the filling quantity can be confirmed by X-ray and CT even in small quantities,
follow-up is easy, and it can be applicable in a wide range of cases. Together, the improvement of
productivity realized by these enhancements has made MTA cement more efficient and economical
than ever before.
These enhancements have been in response to the desire of practitioners using the conventional
product for an MTA cement that is more user-friendly and more broadly applicable, and fromYAMAKIN’s desire for more patients to benefit from the wonderful material that is MTA cement.
We believe that this new Mielle can be used not only by specialists in endodontics, but also bypractitioners who have not used MTA cement before to protect the pulp of numerous patients. We
hope that this book will lead to the correct and widespread use of MTA cement.
The authors

Acknowledgements
The MTA cement project, which led from “TMR-MTA CEMENT” to “Mielle”, was started throughthe enthusiastic commitment of the professors of Health Sciences University of Hokkaido, including
Professor Kazuhiko Endo and Professor Masahito Saito. Subsequent joint research has led to therealization of TMR-MTA CEMENT by conducting compositional studies and evaluations as abiomaterial. Let us take this opportunity to express our heartfelt appreciation to everyone involved.
In order to respond to our clients’ desire to use MTA cement for more patients, we will continue topublicize and widely disseminate information on the correct use of MTA cement through channels
such as this publication.
We also thank many medical professionals, including Professor Testuya Yamamoto, Department ofOral and Maxillofacial Surgery, Kochi Medical School, Kochi University, Dr Mitsuhiko Takata, Takata
Dental Clinic, and Dr Susumu Sudo, Nankodai Dental Clinic, who worked in a variety of ways,
including advice on biological safety assessment, comments from a clinician perspective, and providing
case photographs.
Finally, we would like to express our thanks and heartfelt gratitude to all parties involved in theediting of this publication.

35

Brief History of Supervisors
Dr. Teruo Anraku

Dr. Hidekazu Tanaka

Ph.D. in engineering at KOCHI UNIVERSITY OF TECHNOLOGY

Ph.D. in engineering at KOCHI UNIVERSITY OF TECHNOLOGY

Corporate Technical Advisor, YAMAKIN CO., LTD.

Visiting Researcher, KOCHI UNIVERSITY OF TECHNOLOGY
Chief Researcher, Development Department, YAMAKIN CO., LTD.

Dr. Hiroyuki Itoigawa
Ph.D. in science at HOKKAIDO UNIVERSITY

Dr. Ritaro Matsuura

Academic Technician, Quality Control Section, YAMAKIN CO., LTD.

Ph.D. in Agriculture at United Graduate school of Agricultural Sciences,
Ehime University

Dr. Takahiro Kato

Senior Chief Researcher, Development Department, YAMAKIN CO., LTD.

Ph.D. in engineering at KOCHI UNIVERSITY OF TECHNOLOGY

Dr. Masatoshi Yamazoe

Corporate Officer/ Head Researcher, YAMAKIN CO., LTD.

Ph.D. in dentistry at Matsumoto Dental University

Dr. Takeshi Sakamoto

Part-time lecturer, Matsumoto Dental University and Ishikawa Dental College

Ph.D. in pharmaceutical Science at HOKKAIDO UNIVERSITY

of Dentistry

Corporate Officer/ Technology & Information Marketing Division General

Corporate Officer/ Head Researcher, YAMAKIN CO., LTD.

Manager, YAMAKIN CO., LTD.

Dr. Hirohisa Yamamoto
Dr. Yuji Sato

Ph.D. in entrepreneurial engineering at KOCHI UNIVERSITY OF TECHNOLOGY

Ph.D. in entrepreneurial engineering at KOCHI UNIVERSITY OF TECHNOLOGY

Chairman, YAMAKIN CO., LTD.

Senior Chief Marketing Researcher, Academic Public Relations and IT

Dr. Bunichiro Yamada

Marketing, YAMAKIN CO., LTD.

Ph.D. in engineering, Professor emeritus, Ex-Director, engineering Departmentof Osaka City University
Corporate Advisor, YAMAKIN CO., LTD.

Brief History of authors
Dr. Takahiro Kato
Above-mentioned

Dr. Yuji Sato
Above-mentioned

Mitsuhiko Takata, D.D.S.
Director of TAKATA DENTAL CLINIC, Chuouku, Kobe

Takafumi Nakano
Master in science at HOKKAIDO UNIVERSITY
Resin Development Team, YAMAKIN CO., LTD.

Dr. Ritaro Matsuura
Above-mentioned

Shingo Mizobuchi
Master in science at EHIME UNIVERSITY
Junior Chief Researcher and Project Leader, Organic Material Development Section, YAMAKIN CO., LTD.

MTA CEMENT 2nd Edition

TMR-MTA cement Mielle Product ReportFeatures of TMR-MTA cement Mielle and key points of treatment

Published on July 1, 2019, the 1st Edition
Published August 2, 2019, the 2nd Edition
Publisher: Shigenari Yamamoto
YAMAKIN CO., LTD.
Head Office: 3-7, Sanadayama-cho, Tennoji-ku, Osaka 543-0015, Japan
P: +81-887-55-0281
F: +81-887-55-0018
URL https://www.yamakin-global.com/

Unauthorized reproduction of this book is prohibited, with exceptions under copyright law.

YAMAKIN CO., LTD.

Head Office: 3-7 Sanadayama-cho Tennoji-ku Osaka 543-0015, Japan
Biological Science Safety Laboratory:
Laboratory in the Department of Oral and Maxillofacial Surgery, Kochi Medical School, Kochi University
Kohasu, Oko-cho, Nankoku-shi, Kochi 783-8505, JAPANBranch Office: Tokyo, Osaka, Sendai, Nagoya, Fukuoka, JAPAN
P: +81-887-55-0281 F: +81-887-55-0053
E: contact@yamakin-gold.co.jp

https://www.yamakin-global.com

38

INTERNATIONAL20210122